Project Summary The anti-tumor effects of radiotherapy (RT) are mediated in part by host immune responses,1-4 and the commensal microbiota plays a prominent role in the regulation of immunity.5 Therefore, the overall goal of this proposal is to determine and define the immune cell populations that are altered by the commensal microbiota in order to elucidate how these commensal and immune cells populations alter the anti-tumor actions of RT. Using 16S rDNA sequencing, we will identify commensal candidates that alter the antitumor effects of RT. Our studies will utilize both conventional and germ-free tumor-bearing mice, and we will validate our findings with selective probiotic transfers and antibiotic-treatment during RT to understand how particular commensals regulate the antitumor response. These findings may identify translational opportunities to improve RT by bacterial transfer, probiotics, antibiotics and/or stool transfer. Our preliminary data, generated using both conventional and germ-free tumor-bearing mice, indicate that: 1) Different commensal microbiota produce different anti-tumor responses to RT. 2) The commensal microbiota diminishes anti-tumor response to RT. These results suggest that the eventual clinical translation may be in part mediated by at least the absence of specific microbiota that inhibit anti-tumor responses, or the introduction of microbiota that cultivates a milieu favorable to the antitumor effects of RT. HYPOTHESIS: We hypothesize that the commensal microbiota alters host response to RT, and that it does so through its role in the regulation of host immune status and, consequently, anti-tumor immune response. AIM 1. Determine the effect of microbiota-induced immune cells on RT-mediated antitumor immunity. Goal: To determine and define the immune cell populations that are altered by commensal microbiota in order to elucidate how these populations affect the anti-tumor action of RT. AIM 2. Investigate how particular commensal microbiota members alter RT-response. Goal: To identify the microbiota composition and compare species diversity and microbiota population structure between various groups of RT-treated mice. TRANSFORMATIVE POTENTIAL: This investigation of the microbiota has the potential to change RT practice insofar as the status and effects of commensal microbiota are currently not widely considered as part of standard practice for RT, even though many RT patients currently receive pro- and/or antibiotics prior to or during the course of their treatment, for indications such as treatment toxicity and infection5,28. The long-term goal of our studies is the identification of an optimal microbiota composition for any given tumor phenotype and treatment approach, which would both reduce RT-induced toxicity and potentiate the anti-tumor effects of RT.